Enzymes are remarkable nanomachines that drive cellular processes. They are studied for their fundamental properties to better understand catalysis, and they are studied to improve our ability to design therapeutics against them to benefit human health. After decades of structural biology on enzymes, our understanding of how they work is still growing. Recent work, particularly in the area of NMR spectroscopy, for example, has revealed that dynamic motions and pre-existing conformational switching are critical to the functioning of many, if not all, enzymes. In this proposal, recently developed methods for NMR spin relaxation studies on large proteins, paired with small molecule synthesis and steady-state kinetics, will be applied to a 64 kD enzyme, thymidylate synthase (TS), from E. coli. TS methylates 22-deoxyuridine 52-monophosphate (dUMP) to yield 22- deoxythymidine 52-monophosphate (dTMP), and it is highly conserved from bacteria to humans. Thymidine biosynthesis is a critical metabolic step that, if inhibited, results in cell death. Hence TS is targeted by anticancer drugs and is a viable target for antimicrobials. TS is an obligate homodimer that has a complex, multistep reaction mechanism and displays negative cooperativity. Complex mechanistic features are commonly found in enzymes involved in cell metabolism, yet how these molecules traverse complex energy landscapes to fulfill their function is not well understood. Currently, metabolic enzymes such as TS have not been subjected to thorough analysis of their internal conformational dynamics, largely due to their size. Characterization of TS therefore represents an early example of detailed study of the functional motions of a large metabolic enzyme. The mechanism of TS can be broken down into ~7 distinct steps. Intermediates can be trapped via specific combinations of substrate modifications and mutations, and these intermediates will be studied for their dynamic properties along the reaction coordinate. A key component of the approach will be to not only monitor protein, but to also monitor motions of substrates appropriately labeled for NMR relaxation. Using small molecules as probes will allow active-site motion of all trapped intermediates to be examined in an efficient manner. In aim 1, the effect of substrate binding and bond making/breaking on s-ms and ps-ns motions will be tracked throughout TS using backbone and methyl-based NMR relaxation. In aim 2, the basis for negative cooperativity in TS will be examined from dynamical and steady-state kinetics perspectives. Key to this approach is the generation of mixed asymmetric dimers. In aim 3, previous findings on dihydrofolate reductase (DHFR) will be extended to probe the specific mechanism (and structure) of inhibitor dissociation. A novel relaxation dispersion approach will be taken that will enhance sensitivity to motions that mediate ligand dissociation. Our understanding of enzyme function is growing based on investigation of dynamics in small enzymes. An analogous approach is therefore needed to examine the role of dynamics in larger, more complex enzymes, which should lead to new strategies in drug design and protein engineering.